Nuclear Calcium Concentration Research Articles

Blocking the BKCa channel induces NF-κB nuclear translocation by increasing nuclear calcium concentration†.

Nuclear factor kappa B (NF-κB) transcriptionally regulates several genes involved in initiating uterine contractions. A key factor controlling NF-κB activity is its translocation to the nucleus. In myometrial smooth muscle cells (MSMCs), this translocation can be stimulated by the inflammatory molecule lipopolysaccharide (LPS) or by blocking the potassium calcium-activated channel subfamily M alpha 1 (KCNMA1 or BKCa) with paxilline (PAX). Here, we sought to determine the mechanism by which blocking BKCa causes NF-κB-p65 translocation to the nucleus in MSMCs. We show that LPS- and PAX-induced NF-κB-p65 translocation are similar in that neither depends on several mitogen-activated protein kinase pathways, but both require increased intracellular calcium (Ca2+). However, the nuclear transport inhibitor wheat germ agglutinin prevented NF-κB-p65 nuclear translocation in response to LPS but not in response to PAX. Blocking BKCa located on the plasma membrane resulted in a transient NF-κB-p65 nuclear translocation that was not sufficient to induce expression of its transcriptional target, suggesting a role for intracellular BKCa. We report that BKCa also localizes to the nucleus and that blocking nuclear BKCa results in an increase in nuclear Ca2+ in MSMCs. Together, these data suggest that BKCa localized on the nuclear membrane plays a key role in regulating nuclear Ca2+ and NF-κB-p65 nuclear translocation in MSMCs.

Nuclear localization of NCX: Role in Ca2+ handling and pathophysiological implications

Numerous lines of evidence indicate that nuclear calcium concentration ([Ca2+]n) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca2+ ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na+/Ca2+ exchanger (NCX) isoform 1 seems to play an important role in handling Ca2+ through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca2+ flowing from nucleoplasm into NE lumen thanks to the Na+ gradient created by the juxtaposed Na+/K+-ATPase. Such transfer reduces abnormal elevation of [Ca2+]n within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction.

Calcium Signals in the Plant Nucleus: Origin and Function.

The universality of calcium as an intracellular messenger depends on the dynamics of its spatial and temporal release from calcium stores. Accumulating evidence over the past two decades supports an essential role for nuclear calcium signalling in the transduction of specific stimuli into cellular responses. This review focusses on mechanisms underpinning changes in nuclear calcium concentrations and discusses what is known so far, about the origin of the nuclear calcium signals identified, primarily in the context of microbial symbioses and abiotic stresses.

Inositol 1, 4, 5-trisphosphate-dependent nuclear calcium signals regulate angiogenesis and cell motility in triple negative breast cancer.

Increases in nuclear calcium concentration generate specific biological outcomes that differ from those resulting from increased cytoplasmic calcium. Nuclear calcium effects on tumor cell proliferation are widely appreciated; nevertheless, its involvement in other steps of tumor progression is not well understood. Therefore, we evaluated whether nuclear calcium is essential in other additional stages of tumor progression, including key steps associated with the formation of the primary tumor or with the metastatic cascade. We found that nuclear calcium buffering impaired 4T1 triple negative breast cancer growth not just by decreasing tumor cell proliferation, but also by enhancing tumor necrosis. Moreover, nuclear calcium regulates tumor angiogenesis through a mechanism that involves the upregulation of the anti-angiogenic C-X-C motif chemokine 10 (CXCL10-IP10). In addition, nuclear calcium buffering regulates breast tumor cell motility, culminating in less cell invasion, likely due to enhanced vinculin expression, a focal adhesion structural protein. Together, our results show that nuclear calcium is essential for triple breast cancer angiogenesis and cell migration and can be considered as a promising strategic target for triple negative breast cancer therapy.

CYCLOPS, A DNA-Binding Transcriptional Activator, Orchestrates Symbiotic Root Nodule Development

Nuclear calcium oscillations are a hallmark of symbiotically stimulated plant root cells. Activation of the central nuclear decoder, calcium- and calmodulin-dependent kinase (CCaMK), triggers the entire symbiotic program including root nodule organogenesis, but the mechanism of signal transduction byCCaMK was unknown. We show that CYCLOPS, a direct phosphorylation substrate of CCaMK, isaDNA-binding transcriptional activator. Two phosphorylated serine residues within the N-terminal negative regulatory domain of CYCLOPS are necessary for its activity. CYCLOPS binds DNA in a sequence-specific and phosphorylation-dependent manner and transactivates the NODULE INCEPTION (NIN) gene. A phosphomimetic version of CYCLOPS was sufficient to trigger root nodule organogenesis in the absence of rhizobia and CCaMK. CYCLOPS thus induces a transcriptional activation cascade, in which NIN and a heterotrimeric NF-Y complex act in hierarchical succession to initiate symbiotic root nodule development.

Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells

Studies performed in animals have highlighted the major role of sphingolipids in regulating the balance between cell proliferation and cell death. Sphingolipids have also been shown to induce cell death in plants via calcium-based signalling pathways but the contribution of free cytosolic and/or nuclear calcium in the overall process has never been evaluated.Here, we show that increase in tobacco BY-2 cells of the endogenous content of Long Chain Bases (LCBs) caused by external application of d-erythro-sphinganine (DHS) is followed by immediate dose-dependent elevations of cellular free calcium concentration within the first minute in the cytosol and 10min later in the nucleus. Cells challenged with DHS enter a death process through apoptotic-like mechanisms. Lanthanum chloride, a general blocker of calcium entry, suppresses the cellular calcium variations and the PCD induced by DHS. Interestingly, dl-2-amino-5-phosphopentanoic acid (AP5) and [(+)-dizocilpine] (MK801), two inhibitors of animal and plant ionotropic glutamate receptors, suppress DHS-induced cell death symptoms by selectively inhibiting the variations of nuclear calcium concentration. The selective action of these compounds demonstrates the crucial role of nuclear calcium signature in controlling DHS-induced cell death in tobacco cells.

Chromatin condensation modulates access and binding of nuclear proteins

The condensation level of chromatin is controlled by epigenetic modifications and associated regulatory factors and changes throughout differentiation and cell cycle progression. To test whether changes of chromatin condensation levels per se affect access and binding of proteins, we used a hypertonic cell treatment. This shift to hyperosmolar medium increased nuclear calcium concentrations and induced a reversible chromatin condensation comparable to the levels in mitosis. However, this condensation was independent of mitotic histone H3 serine 10 phosphorylation. Photobleaching and photoactivation experiments with chromatin proteins—histone H2B-GFP and GFP-HP1α—before and after induced chromatin condensation demonstrated that hypercondensation reduced their dissociation rate and stabilized their chromatin binding. Finally, measuring the distribution of nucleoplasmic proteins in the size range from 30 to 230 kDa, we found that even relatively small proteins like GFP were excluded from highly condensed chromatin in living cells. These results suggest that structural changes in condensed chromatin by themselves affect chromatin access and binding of chromatin proteins independent of regulatory histone modifications.—Martin, R. M., Cardoso, M. C. Chromatin condensation modulates access and binding of nuclear proteins.

Regulation of nuclear calcium concentration.

Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.

Sphingolipid metabolites selectively elicit increases in nuclear calcium concentration in cell suspension cultures and in isolated nuclei of tobacco

Sphingolipids are known to interfere with calcium-based signalling pathways. Here we report that these compounds modulate nuclear calcium signalling in tobacco BY-2 cells. Nuclear protein kinase activity phosphorylated endogenous sphingoid long-chain bases (LCBs), suggesting that LCBs are actively metabolized in the nucleus of tobacco BY-2 cells. The Δ4-unsaturated LCB d- erythro-sphingosine and the saturated LCB d- ribo-phytosphingosine elicited increases in free calcium in the nucleus in a dose-dependent and structure-related manner. However, neither sphingosine-1-phosphate nor C2-ceramide was able to stimulate nuclear calcium changes. N-, N-Dimethyl- d- erythro-sphingosine, a structural analogue of d- erythro-sphingosine, was the most efficient LCB so far tested in eliciting nuclear calcium changes both in intact tobacco BY-2 cells and in isolated nuclei. TRP channel inhibitors prevent the effect of DMS, suggesting that LCBs may activate TRP-like channels located on the inner nuclear membrane Collectively, the obtained data show that nuclei respond to LCBs on their own independently of the cytosolic compartment.

Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos

Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)–mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875–878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193–204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types.

Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells

We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627–2641]. These data suggested that in response to elicitors, Ca 2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca 2+] nuc) in response to elicitors. Two types of elicitors are compared: proteins leading to necrosis including four elicitins and harpin, and non-necrotic elicitors including flagellin (flg22) and two oligosaccharidic elicitors, namely the oligogalacturonides (OGs) and the β-1,3-glucan laminarin. Our data indicate that the proteinaceous elicitors induced a pronounced and sustainable [Ca 2+] nuc elevation, relative to the small effects of oligosaccharidic elicitors. This [Ca 2+] nuc elevation, which seems insufficient to induce cell death, is unlikely to result directly from the diffusion of calcium from the cytosol. The [Ca 2+] nuc rise depends on free cytosolic calcium, IP 3, and active oxygen species (AOS) but is independent of nitric oxide.

Nuclear ATP-Gated Channel

The P2X7 receptor binds adenosine triphosphate (ATP) and conducts calcium ions. P2X7 receptors are localized in nerve terminals in the mammalian hippocampus where they presumably detect extracellular ATP. Atkinson et al. report that the channels are also present in the nuclear envelope of neurons in the rat hippocampus with the ATP-binding region facing the cytoplasm. The function of the channels remains to be explored, but given that changes in nuclear calcium concentrations control rates of transcription of certain genes, one can imagine that the channels could couple changes in gene expression to changes in the concentration of ATP in the cytoplasm. L. Atkinson, C. J. Milligan, N. J. Buckley, J. Deuchars, An ATP-gated ion channel at the cell nucleus. Nature 420 , 42 (2002). [Online Journal]

Transcription Requires a Nuclear Signal

Stimuli that increase intracellular cytosolic calcium concentrations sometimes also stimulate an increase in nuclear calcium concentration. Pusl et al. targeted the calcium-binding protein parvalbumin to the cytosol (with a nuclear export signal) or to the nucleus (with a nuclear localization signal) to selectively inhibit cytosolic or nuclear increases in calcium concentration. They used a chimera between the Gal4 DNA binding domain and the mitogen-activated protein kinase (MAPK) target Elk-1 to stimulate reporter gene expression in the presence of the calcium-buffering parvalbumin. Expression of the nuclear-localized parvalbumin selectively inhibited expression from the reporter gene stimulated by the addition of epidermal growth factor (EGF) to activate the MAPK pathway, whereas parvalbumin in the cytosol did not. Nuclear-localized parvalbumin did not inhibit phosphorylation or nuclear localization of the MAPK extracellular-signal-related kinase 2 (ERK2), nor did it inhibit the phosphorylation or translocation of Elk-1. Treatment of the cells with ionomycin to artificially increase calcium concentrations stimulated Elk-1 phosphorylation and translocation but did not stimulate transcription. Thus, calcium alone cannot substitute for a physiological stimulus supplied by EGF. The authors propose that growth factors stimulate multiple signals that result in transcriptional activation, one of which is an increase in nuclear calcium concentration. T. Pusl, J. J. Wu, T. L. Zimmerman, L. Zhang, B. E. Ehrlizh, M. W. Berchtold, J. B. Hoek, S. J. Karpen, M. H. Nathanson, A. M. Bennett, Epidermal growth factor-mediated activation of the ETS domain transcription factor Elk-1 requires nuclear calcium. J. Biol. Chem. 277 , 27517-27527 (2002). [Abstract] [Full Text]

Nuclear and cytosolic calcium changes in osteoclasts stimulated with ATP and integrin-binding peptide

Cytosolic calcium modulates the activity of osteoclasts, large multinucleate cells that resorb bone. Nuclear events, such as gene transcription, are also calcium-regulated in these cells, and fluorescence imaging has suggested that calcium signals produced by some stimuli are specifically targeted to, or amplified within, osteoclast nuclei. We used two alternative techniques of dye loading to examine the changes of intracellular calcium induced in rat osteoclasts by three stimuli. Osteoclasts loaded with the calcium indicator Fura-2 by the acetoxymethyl (AM) ester technique appeared to display marked nuclear calcium amplification. During stimulation with integrin-binding peptides, ATP, or high extracellular calcium, fluorescence ratios recorded from the nuclei rose higher than did ratios recorded from extranuclear regions. In contrast, nuclear calcium amplification was not observed after AM loading in the presence of the anion transport inhibitor sulfinpyrazone, nor in osteoclasts injected with Fura-2 conjugated to a high MW dextran. In these cells, nuclear fluorescence ratios were equal to the extranuclear values at all times: upon stimulation by an agonist, the nuclear and cytosolic calcium concentrations increased by the same amount. The calcium changes seen in stimulated osteoclasts can no longer be taken as evidence for the general validity of the phenomenon of nuclear calcium amplification.

Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells.

The extracellular matrix protein "elastin" is the major component of elastic fibers present in the arterial wall. Physiological degradation of elastic fibers, enhanced in vascular pathologies, leads to the presence of circulating elastin peptides (EP). EP have been demonstrated to influence cell migration and proliferation. EP also induce, at circulating pathophysiological concentrations (and not below), an endothelium- and NO- dependent vasorelaxation mediated by the 67-kDa subunit of the elastin-laminin receptor. Here, by using the techniques of patch-clamp, spectrofluorimetry and confocal microscopy, we demonstrate that circulating concentrations of EP activate low specificity calcium channels on human umbilical venous endothelial cells, resulting in increase in cytoplasmic and nuclear free calcium concentrations. This action is independent of phosphoinositide metabolism. Furthermore, these effects are inhibited by lactose, an antagonist of the elastin-laminin receptor, and by cytochalasin D, an actin microfilament depolymerizer. These observations suggest that EP-induced signal transduction is mediated by the elastin-laminin receptor via coupling of cytoskeletal actin microfilaments to membrane channels and to the nucleus. Because vascular remodeling and carcinogenesis are accompanied by extracellular matrix modifications involving elastin, the processes here described could play a role in the elastin-laminin receptor-mediated cellular migration, differentiation, proliferation, as in atherogenesis, and metastasis formation.

Nuclear calcium: a key regulator of gene expression.

Through the evolution of multicellular organisms, calcium has emerged as the preferred ion for intracellular signalling. It now occupies a pivotal role in many cell types and nowhere is it more important than in neurons, where it mediates both the relaying and long-term storage of information. The latter is a process that enables learning and memory to be formed and requires the activation of gene expression by calcium signals. Evidence from a number of diverse organisms shows that transcription mediated by the transcription factor CREB is critical for learning and memory. Here we review the features of CREB activation by calcium signals in mammalian cells. In contrast to other transcription factors, its regulation is dependent on an elevation of nuclear calcium concentration, potentially placing this spatially distinct pool of calcium as an important mediator of information storage.

Nuclear and cytosolic calcium levels in NIH 3T3 fibroblasts

The spatial distribution of intracellular calcium in resting NIH 3T3 fibroblasts loaded with Fura-2 has been studied by digital image analysis. Calibration parameters were determined separately for the nucleus and the cytosol to take into account possible differences in the physico-chemical properties of the two compartments and were found not to differ significantly. The apparent resting calcium concentration in these cells was found to be significantly lower in the nucleus than in the cytoplasm; however, this difference appears to be an artefact arising from the presence in the cytoplasm of regions with higher calcium levels. Application of thapsigargin, to block active uptake of calcium into these compartments, substantially eliminated the differences between nuclear and cytosolic calcium concentrations. These observations indicate that nuclear and cytosolic calcium are in equilibrium in the resting fibroblasts and argue against the existence of diffusional barriers between these two compartments.

Mechanism of Ca++ Influx in Cerebral Cortical Nuclei during Hypoxia in the Fetal Guinea Pig. † 1769

Previous studies have shown that there is an increase in intracellular calcium concentration during hypoxia in the newborn and fetal brain. The high-affinity Ca++-ATPase enzyme, located on the outer membrane of the nucleus, has been shown to be a primary mechanism of Ca++ uptake into the nucleus in normoxia. In order to elucidate the mechanisms of regulation of nuclear calcium concentration during hypoxia, the present study investigates the effect of hypoxia on the activity of the high-affinity Ca++-ATPase. Nuclear membranes were prepared from the cortex of normoxic (n=5) and hypoxic(n=3) guinea pig fetuses at term. Tissue hypoxia was confirmed by a decrease in the levels of ATP and phosphocreatine. High-affinity Ca++ -ATPase activity was determined in the presence and absence of Mg++ in a medium containing 20 mM HEPES buffer (pH 7.0), 100 mM KCl, 95 μM CaCl2, 0 or 250 μM MgCl2, 1 mM ouabain, 1 mM ATP and 150 μg of nuclear membrane protein. The total, Mg++-dependent, and Mg++-independent Ca++-ATPase activities were calculated and expressed as nmoles/mg protein/hr. In the hypoxic group the total, Mg++ dependent, and Mg++-independent Ca++-ATPase activity increased compared to the normoxic group: 969.7 ± 79 vs 602.4 ± 90.9,p<0.005; 774.7 ± 147.4 vs 491.5 ± 93.8,p<0.01; and 193.8 ± 14.7 vs 132.8 ± 26,p<0.01. The results demonstrate that in the hypoxic nuclear membranes high-affinity Ca++-ATPase activity is increased, suggesting that the structure of the outer nuclear membrane is altered during hypoxia. Since the nuclear calcium signals control nuclear functions such as gene transcription and DNA synthesis, we speculate that the increased high-affinity Ca++-ATPase activity of the nuclear membrane, leading to an increase in nuclear Ca++ concentration, may be a mechanism of altered gene transcription in the hypoxic brain. (Funded by NIH # HD-20337).

Calcium regulation of gene expression: isn't that spatial?

Calcium regulation of gene expression: isn't that spatial?

Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression.

Calcium entry into neuronal cells through voltage or ligand-gated ion channels triggers neuronal activity-dependent gene expression critical for adaptive changes in the nervous system. Cytoplasmic calcium transients are often accompanied by an increase in the concentration of nuclear calcium, but the functional significance of such spatially distinct calcium signals is unknown. Here we show that gene expression is differentially controlled by nuclear and cytoplasmic calcium signals which enable a single second messenger to generate diverse transcriptional responses. We used nuclear microinjection of a non-diffusible calcium chelator to block increases in nuclear, but not cytoplasmic, calcium concentrations following activation of L-type voltage-gated calcium channels. We showed that increases in nuclear calcium concentration control calcium-activated gene expression mediated by the cyclic-AMP-response element (CRE), and demonstrated that the CRE-binding protein CREB can function as a nuclear calcium-responsive transcription factor. A second signalling pathway, activating transcription through the serum-response element (SRE), is triggered by a rise in cytoplasmic calcium and does not require an increase in nuclear calcium.